The measurement of potassium ion (K+) levels in biological samples, either in vivo or in vitro, is of great interest, particularly given the impact ion levels have on many aspects of homeostasis. For example, regulation of K+ in cellular and extracellular compartments is of central importance in volume homeostasis, fluid transport and neuromuscular signaling. Rapid changes in extracellular space (ECS) K+ concentration ([K+]o) occur in the central nervous system during neural activity, with abnormally sustained and spatially-propagating elevations in [K+]o seen in seizures and spreading depression (Somjen et al. J. Neurophysiol. 53, 1098-1108 (1985); Kager et al. J. Neurophysiol. 84, 495-512 (2000)). Assessment of K+ concentration is particularly challenging in these contexts, both by virtue of being an in vivo measurement, as well as requiring both spatial and temporal real-time K+ concentration determination.
K+-sensitive, double-barreled microelectrodes have been the gold standard for in situ biological K+ measurements (Nicholson J. Neurosci. Methods 48, 199-213 (1993); Sick et al. Stroke 30, 2416-2422 (1999). Although accurate measurement of K+ concentration is possible using microelectrodes, their fabrication is technically challenging and their use involves direct invasion of a single measurement site. A K+-sensing fluorescent dye has been developed for cytoplasmic K+ (Minta et al. J. Biol. Chem. 264, 9449-19457 (1989)), though few applications have been reported because of its low fluorescence and poor K+-to-Na+ selectively.
Chromoionophores, and particularly fluoroionophores have gained great interest in use in detection of ion concentrations. Exemplary of such compounds are those having an ionophore portion, which binds an ion of interest, and a fluorophore portion, which provides a fluorescent signal in response to the ion-binding event. Cryptands are one type of ionophore that has been used to create families of chromoionophores (see, e.g., U.S. Pat. No. 6,211,359). These crown ether ionophores form complexes with cations whose ion radius corresponds to that of the cavity formed by the cryptand (Lehn et al. J. Amer. Chem. Soc., 97:6700-6207 (1975)). Ionic radii of the alkali metals Li, Na, K and Rb are 0.78, 0.98, 1.33 and 1.49 Å, respectively (Izatt et. al Chem Rev 1985, 85, 271-339). It is possible to tailor the ether chains of the crown ether or cryptand to target particular cation.
The chemical attributes required for a chromoionophore for a biological application, such as imaging of K+ concentrations in the ECS of the brain, for example, include bright long-wavelength fluorescence, high K+ sensitivity in the physiological concentration range (e.g., up to about 150 mM, with concentrations of about 40 mM in the brain and 120 mM in the extracellular space of other tissues such as airways/lung), pH insensitivity, rapid response, water solubility, and a high K+-to-Na+ selectivity. Additionally, for in vivo imaging of an ECS, the chromoionophore must be membrane impermeable and non-toxic. To date, and to the best of the inventors' knowledge, none of the known chromoionophores using cryptands possess the attributes required for a biological application, such as medical imaging.
For instance, de Silva et al. (Tetrahedron Lett. 1990, 31, 5193-5196) used diazacryptands having aniline-type nitrogens in the crown ether ring for potassium binding. These diazacryptands suffered from sodium interference while determination of potassium ions in the physiological concentration range.
Masilamani et al (U.S. Pat. Nos. 5,439,828, and 5,162,525) describe the utility of diazacryptand functionalized with fluorescent coumarins. These diazacryptand fluoroionophores showed selective for lithium, sodium and potassium ions depending on their molecular structure. The disadvantage of these compounds included a high pH sensitivity and an excitation wavelength at about 330 nm, which is significantly lower than the targeted excitation wavelength of about 500 nm or greater.
He et al (U.S. Pat. No. 6,211,359; see also He et al. J. Am. Chem. Soc. 125:1468-1469 (2003)) showed use of triazacryptand as an ionophore for potassium measurement covalently linked via an alkyl spacer to a napthilimide chromophore, which in turn was conjugated to cellulose. These compounds of He et al. are water insoluble, and the conjugated compounds bound to cellulose exhibited relatively low K+-to-Na+ selectivity.
There is a great need for development of alternative methods to measure K+ concentration in biological samples under physiological conditions. In particular there is a need for methods to measure K+ concentration in living systems, particularly in the brain ECS, which is the small contiguous space between brain cells where rapid changes in [K+]o occur in normal neural activity and in diseases such as epilepsy. Specifically, there is a need for a K+-sensing compound that provides a long wavelength fluorescent signal, is water soluble, membrane impermeant, and is highly potassium selective, insensitive to sodium concentrations and other biologically-relevant cations or anions, and insensitive to pH in the physiological range. Further, such a K+-sensing fluorescent dye should be non-toxic and suitable for use in vivo for a variety of measurements, such as detection of propagating K+ waves in the brain ECS. The present invention addresses these needs.